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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)
EC Number:
230-426-4
EC Name:
2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)
Cas Number:
7128-64-5
Molecular formula:
C26H26N2O2S
IUPAC Name:
5-tert-butyl-2-[5-(5-tert-butyl-1,3-benzoxazol-2-yl)thiophen-2-yl]-1,3-benzoxazole
Details on test material:
- Physical state: yellow powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, England
- Age at study initiation: four - five weeks
- Weight at study initiation: weight range 126 - 131 g (males) and 115 - 120 g (females)
- Fasting period before study: no
- Housing: Five rats of one sex were held in each polypropylene cage (Pattern RCl from North Kent Plastics Limited) measuring 56 x 38 X 18 cm, with stainless steel mesh floors and lids
- Diet: complete low-fat powdered rodent diet (Spratt's Laboratory Animal Diet No. 2, ad libitum
- Water: public supply, water was supplied to each cage via two polythene bottles with chromium plated sipper-tubes.
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 1981-01-21 To: 1981-04-27 (1981-05-07)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the treated groups, the test substance was incorporated into the powdered diet to provide the nominal concentrations.
The latter were held constant throughout the treatment period. A pre-mix was prepared freshly each week and from this the different dietary concentrations were obtained by serial dilution with further quantities of diet. Homogeneity was achieved by mixing (pre-mix) for ten minutes in a Hobart Model A200, then for 15 minutes (final mix) in either a Gardner Interrupted spiral mixer for dietary concentrations of 10000 or 3000 ppm, or in a Hobart model A200 for the dietary concentration of 1000 ppm. After formulation, the diets were sealed in transparent polythene bags within light-proof polythene bans.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the test substance content of diet mixes representative of those to be used during the study was tested for homogeneity and for stability over a four-week period. Spot checks were performed for the test substance content of diets prepared for each treatment level for Weeks 1, 4, 8 and 13.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
contiuously in feed
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm
Remarks:
corresponding to 71.8 mg/kg in males and 78.3 mg/kg in females
Dose / conc.:
3 000 ppm
Remarks:
corresponding to 216.4 mg/kg in males and 246.0 mg/kg in females
Dose / conc.:
10 000 ppm
Remarks:
corresponding to 707.7 mg/kg in males and 788.8 mg/kg in females
No. of animals per sex per dose:
20 male and 20 female rats in each group
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/MORTALITY: Yes
- Time schedule: once or twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day that treatment commenced and at weekly intervals thereafter.
- Group mean values were calculated at the same intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Quantity of food eaten by each cage of rats was calculated weekly by measurement of the amount of food given and that remaining in the food hoppers, together with an estimation of food scattered.

FOOD EFFICIENCY: Yes
- Weekly food conversion ratios, i.e. the weights of food consumed per unit gain in bodyweight, were calculated.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily visual observation of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No / No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before before commencement of treatment, 6 and 12 weeks of treatment
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: obtained from ten male and ten female rats per group
- Parameters checked: Haemoglobin concentration (Hb), Erythrocyte count (RBC), Leucocyte count (WBC) - total, differential, Platelet count, Prothrombin time (PT), Activated partial thromboplastin time (PTTK), Mean cell volume (MCV), Mean cell haemoglobin content (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before commencement of treatment, 6 and 12 weeks of treatment
- Animals fasted: No data
- How many animals: obtained from ten male and ten female rats per group
- Parameters checked: Urea concentration, Glucose concentration, Total protein concentration, Electrophoretic protein fractions, Alkaline phosphatase activity, Alanine amino-transferase activity (ALT), Aspartate amino-transferase activity (AST), Total bilirubin concentration, Direct bilirubin concentration, Total cholesterol concentration, Sodium (Na) and Potassium (K) concentrations, Calcium concentration (Ca), Chloride concentration (Cl), Creatinine concentration, Phosphorus (inorganic) concentration (P)

URINALYSIS: Yes
- Time schedule for collection of urine: before commencement of treatment, 6 and 12 weeks of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Appearance, Volume, pH, Specific gravity (SG), Protein, Total reducing substances, Glucose, Ketones, Bile pigments, Urobilin, Blood, Microscopy - the sediment from centrifugation (3000 rpm)

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

- Terminal observations: All rats surviving until the end of the treatment period and the rat sacrificed prematurely were subjected to euthanasia by carbon dioxide inhalation. Necropsy followed with the minimum of delay.

- The external features of each animal were scrutinised and compared to any relevant comment on the clinical history report. The eyes complete with optic nerve and relevant adnexa were removed. The cranial cap was lifted and the brain dissected free of meninges. The pituitary was freed from the sella turcica and fixed separately. The ventral abdominal skin was reflected to allow observation of the subcutaneous structures, in particular, mammary glands and superficial lymph nodes. Abdominal and thoracic viscera were examined in situ and a note was made of any abnormal position, morphology or interactions. The urinary bladder was slightly inflated with fixative and the urethra ligated. After removal, the bladder was viewed by transmitted light. The mucosa was examined after fixation at the time of embedding. The entire intestinal tract was re-examined after removal. The stomach was opened along its greater curvature and rinsed with isotonic saline, prior to fixation. The caecum was similarly treated. After weighing, both kidneys and the liver were repeatedly sectioned at intervals of a few millimetres; the cut surfaces were examined. The lungs were slightly inflated with fixative via the trachea prior to immersion fixation. The spinal cord was fixed within six thoraco-lumbar vertebrae.

- Organ weight analysis (without connective tissue):
Adrenal glands, Brain, Heart, Kidneys, Liver, Lungs, Ovaries, Pituitary gland, Prostate gland, Spleen, Testes (with epididymides attached), Thymus gland, Thyroid glands, Uterus

HISTOPATHOLOGY: Yes
- Tissues preserved in fixative:
Adrenal glands, Bone, Brain, Caecum, Colon, Duodenum, Eyes and optic nerves, Extra-orbital lacrimal glands, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (including bronchi), Lymph nodes (cervical and mesenteric), Mammary gland (caudal), Oesophagus, Ovaries, Pancreas, Pituitary gland, Prostate gland, Salivary gland (left), Seminal vesicles, Skeletal muscle, Skin, Spleen, Stomach (fundus and pylorus), Testes, Thymus gland, Thyroid glands, Trachea, Urinary bladder, Uterine cervix, Uterus

- Other tissues preserved (possible future requirement for microscopic evaluation):
Aorta, Mammary gland (cranial), Sciatic nerve (left), Spinal cord, Salivary glands (right), Tongue,

- Microscopic examination: From all of the control rats, and those rats given the highest dosage of the test substance, all the tissues listed above were subjected to microscopic examination. From rats receiving the low or intermediate dosage of the test substance, the heart, liver, kidneys and macroscopic abnormalities were subjected to microscopic examination. After dehydration and embedding in paraffin wax, sections of the required tissues were cut at 5 pm thickness and stained with haematoxylin and eosin. Both glandular and non-glandular areas of the stomach, and both auricular and ventricular sections of the heart, were thus prepared.
The brain was sectioned at three levels (cerebellum, cerebral cortex and medulla). Two sections each of the liver, lungs and lymphatic tissue were prepared.
Other examinations:
Concentration measurements of test substance in dietary samples confirmed the nominal test substance concentrations.
Statistics:
Statistical evaluation, by Student's t-test using a pooled within-group error variance, has been performed on the following:
- Overall food intake for selected periods
- Bodyweight increment for selected periods
- Haematology
- Blood chemistry
- Absolute organ weights
- Bodyweight-relative organ weights.
[Unless stated, group mean values were not significantly different from controls (p > 0.05)].

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs of reaction to treatment or deaths which could be ascribed to the administration of the test substance.
Mortality:
no mortality observed
Description (incidence):
One male rat receiving 3000 ppm was killed on humane grounds during Week 6 due to respiratory distress. Breathing appeared to be impaired by bleeding from a wound inside the upper lip and the resultant swelling of the muzzle. Necropsy did not reveal any treatment-related changes.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The bodyweight gain of rats receiving the test substance was unaffected by treatment.
Occasional inter-group differences in weight gain achieved statistical significance (p < 0.05), but these lacked a consistent pattern among the treated groups and were not dosage-related. They were, therefore, not considered to be of any biological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of treated rats was similar to that of the controls throughout the treatment period. The food consumption of females receiving 1000 ppm was marginally lower than that of the controls, but this was considered fortuitous and of no biological significance.
Food efficiency:
no effects observed
Description (incidence and severity):
The efficiency of food utilisation of treated rats, as estimated from their food conversion ratios, was broadly similar to that of the controls. The slightly higher overall food conversion ratio for females receiving 3000 ppm mainly reflected the slightly inferior weight gain of this group and was not attributed to treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily examination of water bottles did not reveal any differences in the water consumption of treated and control groups.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological examination after six and 12 weeks of treatment did not reveal any effects that could be ascribed to treatment.
Such statistically significant inter-group differences as were detected lacked dosage-relationship and were not apparent on both occasions of examination.
After 12 weeks of treatment, low erythrocytic characteristics, packed cell volume, haemoglobin concentration and erythrocytic count in rat contributed to lower group mean values for these characteristics in males receiving 3000 ppm, when compared with control males. The resulting inter-group differences were not considered to be related to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical examination of plasma after six and 12 weeks of treatment did not reveal any effects that could be attributed to treatment. Such inter-group differences as were detected generally lacked a consistent trend or dosage-relationship, were not apparent on both occasions of examination and although in some cases attained statistical significance, were generally too small in magnitude to be of biological significance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in the chemical characteristics or constituents of urine examined that could be ascribed to treatment with the test substance. After 11 weeks of treatment marginally lower urine volumes recorded in treated rats were attributable to high values recorded in some animals of the control group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and body weight-relative liver weights of males and females receiving 10000 ppm were slightly higher than those of control animals.
There was no other inter-group difference in organ weights that could be ascribed to treatment. Higher body weight-relative liver weights in female rats receiving 1000 or 3000 ppm were attributed to the minimally lower body weight of these animals in comparison with control females, and were not considered to be of biological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no changes which associated with treatment in animals killed at termination. A range of commonplace lesions was present in the animals examined; such changes included subpleural foci and petechiae on the lungs of a few animals in control or treated groups. Slight hydronephrosis was present in a small number of male rats in the control or treated groups.
In one female animal in the lowest dosage group calculi were present in the urogenital tract. These findings are considered incidental.
In the male of Group 3 which was killed on humane grounds, a small range of commonplace lesions was present; these included pulmonary petechiae and pallor, prominent parathyroids, and enlarged and occasionally congested cervical lymph nodes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Animals killed at termination of the study period: There were no pathological findings associated with treatment with the test substance.
(There was a range of mundane degenerative and inflammatory findings present in the tissues examined, similar in type and incidence to those considered usual in CD rats at this laboratory)
Animal killed in extremis: One male rat receiving 3000 ppm was killed on humane grounds. There were no pathological findings associated with treatment with the test substance. The cause of the terminal condition in this animal was considered to be acute periodontitis with intraepidermal oedema and haemorrhage.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Achieved dosages, expressed as mg/kg/day, fell as the treatment period progressed, reflecting the declining ratio between food consumption and body weight. Although there were some inter-group differences in respect of growth and food consumption, the relationship between treatment levels was generally maintained in terms of the achieved dosage.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 000 other: ppm (mg/kg food)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOEL
Effect level:
3 000 other: ppm (mg/kg food)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects noted

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Achieved Doses:

(mg/kg body weight)

Male Animals Female Animals
Week 1000 ppm 3000 ppm 10000 ppm 1000 ppm 3000 ppm 10000 ppm
1 136.2 421.8 1329 130.6 398.9 1274
2 111.9 337.3 1119 109.5 339.4 1102
3 95.3 289.2 943 96.6 301.2 991
4 83.7 253.6 832 91.4 277.7 919
5 73.3 218 735 82.5 252.9 837
6 65.5 198.5 647 75.6 237.8 780
7 58.7 169.9 569 67.1 214.1 670
8 55.3 166.9 543 65.6 220.9 666
9 55.4 159.5 524 63.9 205 634
10 51.3 151.8 492 60.5 192.9 617
11 49.3 151.5 512 61 192.3 605
12 47.7 146.7 471 58.4 186.3 594
13 49.4 147.9 484 55.1 178.5 565
Average 71.8 216.4 707.7 78.3 246.0 788.8

Applicant's summary and conclusion

Conclusions:
The only changes that could confidently be ascribed to treatment with the test substance were slightly higher absolute and body weight-relative liver weights of rats receiving 10000 ppm and even these inter-group differences from controls were minimal. The liver weights of rats receiving 3000 ppm were not disturbed by treatment. There was no evidence of histopathological change in the liver or the other tissues examined. It is concluded therefore that the test substance at concentrations of 1000 and 3000 ppm is without toxicological effect in dietary administration to the rat.
Executive summary:

Groups of 20 male and 20 female CD rats were fed diets containing the test article at concentrations of 1000, 3000 or 10,000 ppm for 13 weeks. A similarly constituted group of rats received untreated diet and served as a control group. There were no signs of reaction to treatment. One male receiving 3000 ppm was killed on humane grounds; necropsy and microscopic examination of the tissues did not reveal any lesions that could be ascribed to treatment. Food and water consumption and body weight gain were unaffected by administration of test material. Food conversion ratios of the treated rats remained broadly similar to those of the controls throughout the treatment period. Haematological examination after six and 12 weeks of treatment did not reveal any changes that could be attributed to treatment. Examination of the chemical composition of the plasma after six and 12 weeks of treatment did not reveal any effects that could be attributed to treatment. Urinalysis after six and 11 weeks did not reveal any treatmentrelated effects. Macroscopic examination of the tissues of rats killed at termination did not reveal any changes that could be ascribed to treatment. The absolute and bodyweight-relative liver weights of rats receiving 10,000 ppm were slightly higher than those of controls. Microscopic examination of the tissues of rats killed at termination did not reveal any changes that could be ascribed to treatment. It is concluded that the administration of the test item at a dietary concentration of 3000 ppm elicited no manifestation of toxicity and is considered as the NOEL.